The widespread bacterial phosphoenolpyruvate (PEP):sugar phosphotransferase system (PTS) couples the translocation and phosphorylation of numerous sugars. The system is composed of two cytoplasmic proteins (enzyme I and HPr) that are used for all sugars as well as sugar specific, membrane-bound components known as enzymes II and III. PEP is the phosphoryl donor in the Mg(II)-dependent autophosphorylation of enzyme I on histidine 189. Thermal stabilities of enzyme I (63,562 Da subunit) of the E. coli PTS and a cloned amino terminal domain of enzyme I (EIN; 28,346 Da) have been investigated by differential scanning calorimetry (DSC) and far UV circular dichroism (CD) at pH 7.5. Previously, we showed that phosphorylation of the active-site His 189 destabilizes the amino terminal domain of enzyme I by ca. 7 C. A decrease in the conformational stability of the amino terminal domain by phosphorylation of His 189 promotes phosphotransfer to HPr (the next protein of the PTS). In order to investigate the effects of phosphorylation on the stability of enzyme I further, we have substituted Ala and Glu for His 189 to produce H189A and H189E mutants of both the amino terminal domain (EIN) and the full length enzyme I. DSC and temperature-induced changes in ellipticity at 222 nm for wild-type (H189) and mutant EIN proteins have shown two-state unfolding for all three proteins in 10 mM K-phosphate buffer, pH 7.5, with an unfolding enthalpy of 140 kcal/mol. At low ionic strength, transition temperature (Tm) values were 57.0, 55.4, and 53.3 C for H189, H189A, and H189E, respectively. Thus, the mutations at His 189 decreased the thermal stability of EIN and the introduction of a negative charge at the active site of EIN was the most destabilizing. Neutral salt had the greatest shielding effect on the H189E mutant. The binding of HPr produced a 3 C stabilization in each case. Long-range interactions between the N- and C-terminal domains of intact enzyme I are being investigated. The C-terminal domain is necessary for dimerization of enzyme I and preliminary sedimentation equilibrium studies suggest that the monomer-dimer equilibrium is affected by phosphorylation of His 189 or by substitution of Glu for His at position 189 in the N-terminal domain. DSC results indicate that thermal unfolding and refolding reactions of the N- and C-terminal domains are weakly coupled energetically. - enzyme I, PEP:sugar phosphotransferase system, amino terminal, domain, phosphorylation, stability, calorimetry, circular dichroism